Resolution of the basal plasma membrane calcium flux in vascular smooth muscle cells
Fayazi, A.H.; Lapidot, S.A.; Huang, B.K.; Tucker, R.W.; Phair, R.D.
American Journal of Physiology 270(6 Pt 2): H1972-H1978
1996
ISSN/ISBN: 0002-9513 PMID: 8764246 Document Number: 458122
Steady-state cytosolic calcium (Ca-i-2+) concentration in a vascular smooth muscle cell is determined by Ca-2+ influx and Ca-2+ extrusion across the plasma membrane, yet no means for determining the absolute magnitude of these transmembrane Ca-2+ fluxes in the basal state of the resting cell has been devised. We now report a method that combines fluorescence measurement of Ca-i-2+, 45Ca kinetics, and computer modeling to yield the basal plasma membrane Ca-2+ flux in A7r5 vascular smooth muscle cells. Kinetic analysis of basal Ca-i-2+ and Ca-i-2+ transients following chelation of extracellular Ca-2+ yields a unique value for the ratio of the rate constant governing Ca-2+ pumping into the sarcoplasmic reticulum (SR) to that for plasma membrane Ca-2+ extrusion (1.12 +- 0.06). When this ratio was used to constrain the least-squares fitting of 45Ca efflux data from A7r5 cells, it was possible to determine unique values for the unidirectional, steady-state Ca-2+ fluxes across both SR and plasma membranes. The basal unidirectional plasma membrane Ca-2+ flux was 0.062 +- 0.018 fmol cntdot min-1 cntdot cell-1, and the basal SR Ca-2+ flux was 0.069 +- 0.019 fmol cntdot min-1 cntdot cell-1. These results demonstrate, within the limitations of measuring the absolute value of Ca-i-2+, the feasibility of measuring previously unresolvable subpicoamp basal Ca-2+ fluxes in intact cells under normal physiological conditions.